Graduation Year

2025

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Thomas Taylor-Clark, Ph.D.

Committee Member

Paula Bickford, Ph.D.

Committee Member

Jerome Breslin, Ph.D.

Committee Member

Jay Dean, Ph.D.

Committee Member

Sami Noujaim, Ph.D.

Committee Member

Donald Bolser, Ph.D.

Keywords

Sensory neurobiology, Respiratory physiology, Autonomic function, TRPV1, TRPA1

Abstract

The sensory nerves in the airways directly regulate critical defenses and indirectly regulate vital gas exchange. Airway function is largely controlled by the autonomic nervous system, and our understanding of its sensory regulation is directly connected to our ability to treat respiratory and cardiovascular pathologies. Airway sensory afferents are heterogeneous with respect to their electrophysiological properties, peptidergic capacity, and stimuli sensitivity, which at least in part dictate some of their various functions. There is evidence that embryological origin impacts reflex function: activation of the neural crest-derived population of sensory afferents has been shown to evoke the cough reflex whereas activation of the placode-derived population can inhibit it (Chou et al., 2017). Nociceptors are also sensitive to a wide range of stimuli such as inflammatory mediators, allergens, irritants, and environmental pollutants and are thought to be involved in mediating many symptoms of pulmonary inflammation (Taylor-Clark, 2020). As such, these nerves represent important targets for therapeutic intervention.

Indeed, sensory nerve activation can evoke many reflexes in the cardiopulmonary system that overlap with those seen in pathological cardiovascular and respiratory conditions (Taylor-Clark & Undem, 2022). One question that remains is which sensory afferents evoke which distinct reflexes? The diverse reflexes caused by stimulation of sensory nerves may be explained by afferent heterogeneity, however it is still unknown which parameters define functionally distinct subpopulations of pulmonary afferents. Are these reflexes determined by embryological source, receptor expression, nerve termination patterns (which may or may not be overlapping subsets), or some other unknown parameter? Our goal was to determine which afferent subsets are responsible for the different cardiovascular and respiratory reflexes so that we can target their pathological functions without interrupting their homeostatic functions.

The term “cardiopulmonary reflex” might suggest a relatively simple feedback/feedforward mechanism underlying respiratory and cardiovascular reflexes. This would imply straightforward treatment strategies for its role in autonomic dysfunction. However, careful review of the literature reveals complex neuronal networks whose study have been hindered by practical limitations. Evidence suggests different functions of the airway sensory afferents originating from the nodose and jugular ganglia (the two components of the vagus (Mazzone & Undem, 2016), but the lack of subset-specific agonists has restricted the study of these nerves. Adding additional complexity, functional reflexes require communication between multiple organ systems and as such should be measured with in vivo physiological studies.

In previous studies, selective stimulation of the airways has often been accomplished with the use of anesthesia. However, some of the cardiopulmonary reflexes are sensitive to and altered by anesthesia, which can either make it appear as if there is no reflex response at all or even cause an opposite effect (e.g. allyl isothiocyanate (AITC, TRPA1 agonist) evoking tachypnea instead of bradypnea (Hooper et al., 2016)). Finally, although the vagus nerve is responsible for the majority of sensory innervation in the airways, the dorsal root ganglion (DRG) and trigeminal nerves are also found in the airways, further complicating selective activation of afferent subsets. The conceptual innovation of the following experiments is the identification of specific afferent subsets responsible for reflex regulation.

Our lab has combined the use of several approaches to circumvent the limitations described above. Firstly, we use radiotelemetry and whole-body plethysmography to measure heart rate and respiration in awake and freely-moving animals. The ability to measure these reflexes without the use of anesthesia is critical to uncovering the functions of these afferent subsets in healthy physiological conditions. Secondly, to address the issue of selectivity, we have combined the following techniques: chemogenetics, adeno-associated viruses (AAVs), vagal ganglia injections, and intersectional genetics. To compensate for the lack of subset-specific agonist, we use the chemogenetic tool DREADD (Designer-Receptor Exclusively Activated by Designer Drug), allowing for recombinase-driven insertion of a modified muscarinic receptor (activated only by the otherwise inert clozapine-N-oxide, CNO), into our population of interest. The jugular DREADD mouse models we have developed are some of the first to demonstrate in vivo reflex regulation by jugular-specific activation in awake mice. Then, to account for the potential influence of DREADD expression in non-vagal afferents, we performed vagal injections of AAVs for Cre-driven selective insertion of DREADD receptors into only the vagal afferents. Finally, we incorporated intersectional genetics (Fenno et al., 2014): by manipulating the distinct genetically-defined subpopulations (combinations of TRPV1+/-, peptidergic+/-, nodose +/-), we can determine the functions of nociceptive and non-nociceptive afferent subsets. By systematically identifying the functions of these sensory nerves, we seek to identify selective therapeutic targets that will facilitate better treatments for respiratory and cardiovascular disease.

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